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April 21, 2020  |  

Development of Tigecycline Resistance in Carbapenemase-Producing Klebsiella pneumoniae Sequence Type 147 via AcrAB Overproduction Mediated by Replacement of the ramA Promoter.

Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates.The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine ß-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR.Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene.The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement. © The Korean Society for Laboratory Medicine.

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April 21, 2020  |  

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected.To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms.The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing.A novel IncZ-7 plasmid pEC013 (~118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013.The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid’s heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020  |  

Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai, China, 2006-2016.

Colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella. Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1-harbouring Salmonella in community-acquired infections should be estimated.This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006-2016. Molecular characteristics of the mcr-1-positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes.Among the 12,053 Salmonella isolates, 37 mcr-1-harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1-harbouring strains were aged <5?years. All strains, including fluoroquinolone-resistant and/or extended-spectrum ß-lactamase-producing strains, were multi-drug resistant. S. Typhimurium had higher mcr-1 plasmid acquisition ability compared with other common serovars. Phylogeny based on the genomes combined with complete plasmid sequences revealed some clusters, suggesting the presence of mcr-1-harbouring Salmonella outbreaks in the community. Most mcr-1-positive strains were clustered together with the pork strains, strongly suggesting pork consumption as a main infection source.The mcr-1-harbouring Salmonella prevalence in community-acquired diarrhoea displays a rapid increase trend, and the ESBL-mcr-1-harbouring Salmonella poses a threat for children. These findings highlight the necessary and significance of prohibiting colistin use in animals and continuous monitoring of mcr-1-harbouring Salmonella.Copyright © 2019. Published by Elsevier B.V.

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April 21, 2020  |  

A novel plasmid carrying carbapenem-resistant gene blaKPC-2 in Pseudomonas aeruginosa.

A carbapenem-resistant Pseudomonas aeruginosa strain PA1011 (ST463) was isolated from a patient in a surgical intensive care unit. PCR detection showed that PA1011 carried the blaKPC-2 gene. A plasmid was isolated and sequenced using the Illumina NextSeq 500 and PacBio RSII sequencing platforms. The plasmid was named pPA1011 and carried the carbapenem-resistant gene blaKPC-2. pPA1011 was a 62,793 bp in length with an average G+C content of 58.8%. It was identified as a novel plasmid and encoded a novel genetic environment of blaKPC-2 gene (?IS6-Tn3-ISKpn8-blaKPC-2-ISKpn6-IS26).

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April 21, 2020  |  

Whole genome assembly and functional portrait of hypervirulent extensively drug-resistant NDM-1 and KPC-2 co-producing Klebsiella pneumoniae of capsular serotype K2 and ST86.

To characterize an emergent carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strain, NUHL30457, which co-produces NDM-1 and KPC-2 carbapenemases.We performed WGS analysis on a clinical carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) strain NUHL30457. Sequence data were analysed using comparative genomics and phylogenetics. WGS was used to perform MLST, capsular genotyping and identification of virulence and antimicrobial resistance genes. The virulence of NUHL30457 was analysed by serum killing assay, neutrophil phagocytosis and mouse lethality assay.The NUHL30457 strain was carbapenem resistant and belonged to ST86 and serotype K2. A significant increase in resistance to serum killing and antiphagocytosis was found in the NUHL30457 strain compared with the reference strain. The murine lethality assay showed an LD50 of 2.5?×?102?cfu for the NUHL30457 strain, indicating hypervirulence. WGS revealed that NUHL30457 has a single 5.3?Mb chromosome (57.53% G?+?C content) and four plasmids in the range 49.2-215.7?kb. The incompatibility group (Inc)N plasmid p30457-4 carried the blaNDM-1 and qnrS1 genes. The IncFII(K) plasmid p30457-3 also carried an array of resistance elements, including blaCTX-M-65, blaTEM-1 and blaKPC-2. The IncHI1/IncFIB plasmid p30457-1, which carried virulence genes, was identical to a pLVPK plasmid reported previously.To the best of our knowledge, this is the first report to isolate an ST86 hvKP strain that co-produces NDM-1 and KPC-2 carbapenemase. Further investigation is required to reinforce our understanding of the epidemiology and virulence mechanisms of this clinically significant CP-hvKP. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020  |  

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Plasmid analysis of Escherichia coli isolates from South Korea co-producing NDM-5 and OXA-181 carbapenemases.

Recently, Escherichia coli isolates co-producing New Delhi metallo-ß-lactamase (NDM)-5 and oxacillinase (OXA)-181 were identified in a tertiary-care hospital of South Korea. Isolate CC1702-1 was collected from urine in January 2017 and isolate CC1706-1 was recovered from a transtracheal aspirate of a hospitalized patient in May 2017. Carbapenemase genes were identified by multiplex PCR and sequencing, and whole genome sequencing was performed subsequently using the PacBio RSII system. Both E. coli isolates belonged to the same clone (ST410) and were resistant to all ß-lactams including carbapenems. We obtained whole plasmid sequences of the isolates: pCC1702-NDM-5 from CC1702-1 and pCC1706-NDM-5 and pCC1706-OXA-181 from CC1706-1. The two E. coli isolates belonged to the same clone (ST410) and they were completely resistant to all ß-lactams, as well as carbapenems. Two blaNDM-5-harboring plasmids belonged to the same incompatibility group, IncFIA/B, and consisted of 79,613?bp and 111,890?bp with 87 and 130 coding sequences, respectively. The genetic structures of the two blaNDM-5-bearing plasmids, which were distinct from the blaNDM-5-bearing plasmids from the Klebsiella pneumoniae isolates previously transmitted from the United Arab Emirates (UAE) to South Korea, differed from each other. While pCC1702-NDM-5 showed high degree of identity with the plasmid from a multidrug-resistant isolate of Citrobacter fruendii P5571 found in China, pCC1706-NDM-5 was very similar to the plasmid from a multidrug-resistant isolate of E. coli AMA1176 found in Denmark. pCC1706-OXA-181, which was a 51?kb, self-transmissible IncX3 plasmid, was identical to the E. coli plasmids pAMA1167-OXA-181 from Denmark and pOXA-181-WCHEC14828 from China. Plasmids harboring blaNDM-5 in E. coli isolates might not be transferred from K. pneumoniae isolates co-producing NDM-5 and OXA-181. They probably originated from multiple sources.Copyright © 2019 Elsevier Inc. All rights reserved.

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April 21, 2020  |  

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020  |  

Sequencing and Genomic Diversity Analysis of IncHI5 Plasmids.

IncHI plasmids could be divided into five different subgroups IncHI1-5. In this study, the complete nucleotide sequences of seven blaIMP- or blaVIM-carrying IncHI5 plasmids from Klebsiella pneumoniae, K. quasipneumoniae, and K. variicola were determined and compared in detail with all the other four available sequenced IncHI5 plasmids. These plasmids carried conserved IncHI5 backbones composed of repHI5B and a repFIB-like gene (replication), parABC (partition), and tra1 (conjugal transfer). Integration of a number of accessory modules, through horizontal gene transfer, at various sites of IncHI5 backbones resulted in various deletions of surrounding backbone regions and thus considerable diversification of IncHI5 backbones. Among the accessory modules were three kinds of resistance accessory modules, namely Tn10 and two antibiotic resistance islands designated ARI-A and ARI-B. These two islands, inserted at two different fixed sites (one island was at one site and the other was at a different site) of IncHI5 backbones, were derived from the prototype Tn3-family transposons Tn1696 and Tn6535, respectively, and could be further discriminated as various intact transposons and transposon-like structures. The ARI-A or ARI-B islands from different IncHI5 plasmids carried distinct profiles of antimicrobial resistance markers and associated mobile elements, and complex events of transposition and homologous recombination accounted for assembly of these islands. The carbapenemase genes blaIMP-4, blaIMP-38 and blaVIM-1 were identified within various class 1 integrons from ARI-A or ARI-B of the seven plasmids sequenced in this study. Data presented here would provide a deeper insight into diversification and evolution history of IncHI5 plasmids.

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April 21, 2020  |  

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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April 21, 2020  |  

Complete genome sequence of acetate-producing Klebsiella pneumoniae L5-2 isolated from infant feces.

Acetate is an important metabolite in infants as it can affect metabolism as well as immune and inflammatory responses. However, there have been no studies on acetate production by Klebsiella pneumoniae isolated from infant feces. In this study, we isolated a K. pneumoniae strain, L5-2, from infant feces, and we found it produces acetate. The genome of L5-2 consisted of a 5,237,123-bp single chromosome and a 139,211-bp single plasmid. The G?+?C content was 57.27%. By whole-genome analysis of K. pneumoniae L5-2, we identified seven genes related to acetate production (poxA, pta, eutD, ackA, eutP, eutQ, and adhE). We confirmed acetate production by K. pneumoniae L5-2 by ion chromatography. The aldehyde/alcohol dehydrogenase (adhE) activity of K. pneumoniae L5-2 was significantly higher than that of the K. pneumoniae subsp. ozaenae ATCC 11296. Thus, the acetate-producing ability of K. pneumoniae L5-2 was influenced by the adhE gene. In addition, K. pneumoniae L5-2 had significantly less virulence factor-encoding genes than other K. pneumoniae strains isolated from humans. In conclusion, K. pneumoniae L5-2 isolated from infant feces has less virulence factors and higher adhE activity than other K. pneumoniae strains.

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April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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April 21, 2020  |  

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241?bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2?)-Ia, two Tn402-like with ant(3?)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

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April 21, 2020  |  

Whole-genome sequencing of Klebsiella pneumoniae isolates to track strain progression in a single patient with recurrent urinary tract infection.

Klebsiella pneumoniae is an important uropathogen that increasingly harbors broad-spectrum antibiotic resistance determinants. Evidence suggests that some same-strain recurrences in women with frequent urinary tract infections (UTIs) may emanate from a persistent intravesicular reservoir. Our objective was to analyze K. pneumoniae isolates collected over weeks from multiple body sites of a single patient with recurrent UTI in order to track ordered strain progression across body sites, as has been employed across patients in outbreak settings. Whole-genome sequencing of 26 K. pneumoniae isolates was performed utilizing the Illumina platform. PacBio sequencing was used to create a refined reference genome of the original urinary isolate (TOP52). Sequence variation was evaluated by comparing the 26 isolate sequences to the reference genome sequence. Whole-genome sequencing of the K. pneumoniae isolates from six different body sites of this patient with recurrent UTI demonstrated 100% chromosomal sequence identity of the isolates, with only a small P2 plasmid deletion in a minority of isolates. No single nucleotide variants were detected. The complete absence of single-nucleotide variants from 26 K. pneumoniae isolates from multiple body sites collected over weeks from a patient with recurrent UTI suggests that, unlike in an outbreak situation with strains collected from numerous patients, other methods are necessary to discern strain progression within a single host over a relatively short time frame.

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September 22, 2019  |  

Rapid infectious disease identification by next-generation DNA sequencing.

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

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